Lignin, a major component of cell walls, is the third most-abundant biopolymer and the largest available resource of natural aromatic polymers. It is mainly composed of the monolignols p-coumaryl, coniferyl, and sinapyl alcohols which give rise to the p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) lignin units (e.g., Bocrjan et al, Annual Review of Plant Biology 54:519-546, 2003). Unfortunately, it is also the primary contributor to the high cost of lignocellulosic sugar production, because cell wall polysaccharides are encrusted with lignin, which make them highly resistant to extraction and enzymatic hydrolysis. Moreover, lignin has almost no commercial value aside from its role as a source of heat, and it is generally treated as a waste product.
Lignin has been a target of genetic manipulation for several decades because its content in biomass is inversely correlated with its forage digestibility and kappa value in the pulping industry. Lignin biosynthesis is well-characterized and all the enzymes required for the synthesis of its three major building blocks—called monolignols—are well-known and highly-conserved in all vascular plants. However, lignin cannot be readily removed from growing plants without causing deleterious developmental effects (e.g., Bonawitz & Chapple, Curr Opin Biotechnol 24:336-343, 2013). Genetic manipulation trials using natural mutants or silencing strategies have failed because they drastically reduced lignin content in a non-selective way. Although there are cases in which mild genetic manipulations have been used to moderately reduce lignin content or modify its composition in biomass, modestly improving saccharification efficiency, forage digestibility, and pulping yield (e.g., Li et al., Plant Journal 54:569-581, 2008), these approaches are still rather limited.
Classical lignin-modification methods typically repress the expression or activity of lignin biosynthetic genes. They require identification of natural defective alleles, the screening of single-nucleotide polymorphisms (SNPs) from mutant populations (usually a labor-intensive process) or the development of RNAi-based gene-silencing approaches. A limitation to these approaches is the lack of tissue specificity because every cell carries the same defective allele or silenced gene because RNAi moves from cell-to-cell and affect most of the tissues in the plant (Brosnan & Voinnet, Curr Opin Plant Biol 14:580-587, 2011). Moreover, they affect not only the lignin biosynthesis pathway, but also have indirect effects on other metabolic routes connected to the phenylpropanoid and monolignol pathways. The phenylpropanoid pathway, for example, generates a wide array of secondary metabolites that contribute to all aspects of plant development and plant responses to biotic and abiotic stresses (e.g., Vogt, Molecular Plant 3:2-20, 2010).
There is a need for new methods to reduce lignin content further, without altering plant development or causing undesirable effects. This invention addresses that need.